The hormonal form of vitamin D, calcitriol, acts through the vitamin D receptor (VDR) to regulate important functions such as calcium homeostasis, cell proliferation, differentiation, and immune functions. Defects in the target actions of calcitriol thus have a broad spectrum of manifestations ranging from hyperreactivity to a lack of effects. Hereditary resistance to calcitriol usually results from a mutation in the VDR gene and manifests as rickets. We used skin fibroblasts from subjects with this rare hereditary disease to explore mechanisms of calcitriol action. These mutant cells display abnormalities in many discrete steps of the receptor activation pathway. They have been used to establish that certain receptor mutations compromise many receptor functions but allow other functions to be retained normally. We used these natural "knockout" cells to show that a nongenomic action of calcitriol to raise intracellular cGMP level is mediated through the VDR. To study VDR localization in living cells, we developed the first biologically active fluorescent ligand for calcitriol: 38-BODIPY-calcitriol. This labeled hormone made possible the use of confocal microscopy to show that VDR reside both in the cytoplasm and the nucleus, and that calcitriol exposure induces translocation of cytoplasmic VDR into the nucleus. We identified a docking site for VDR in the endoplasmic reticulum, bound to calreticulin. Experiments in living cells also showed that VDR associates with microtubules during translocation, and this finding was confirmed by in vitro copolymerization studies with adenovirus expressed VDR and purified tubulin. Fluorescent labeling techniques proved useful in elucidating the mechanisms of VDR activation and led us to understand better abnormalities in the vitamin D effector system. A transcriptionally active chimeric protein was prepared in Dr. Hager's laboratory, containing the green fluorescent protein and the glucocorticoid receptor (GFP-GR). Transient expression of GFP-GR in mouse adenocarcinoma cells allowed us to study receptor motion and intranuclear targeting. We confirmed that unliganded GR resides in the cytoplasm and that hormone addition initiates a cytoplasm to nucleus translocation. We detected distinct subnuclear localizations of agonist (dexamethasone) and antagonist (RU486) activated receptors. After dexamethasone, GFP-GR accumulated in a discrete series of foci, while after RU486, GFP-GR remained in a reticular pattern, not forming bright foci. Another adenocarcinoma cell line (3134) was used to define the nature of these foci. These cells carry a Aminichromosome@ containing 800 GR binding sites in a head-to-tail tandem repeat. Agonist exposure caused GFP-GR accumulation along this array. A mutant GFP-GR, with altered DNA-binding failed to localize to this array and failed to activate transcription of this Aminichromosome. We prepared green fluorescent protein VDR chimeras to study translocation and intranuclear distribution of VDR. Transient expression of this GFP-VDR in mouse adenocarcinoma and in COS-7 cells showed that the chimeric protein is competent for hormone dependent transactivation and confirmed that hormone induces translocation of cytoplasmic VDR into the nucleus. Green fluorescent protein chimeras of mutant VDR variants are being used to gain further understanding of the mechanisms of vitamin D resistant rickets.